Treacher Collins syndrome (TCS) is an autosomal dominant, craniofacial disorder. It is characterized by mandibulofacial dysostosis with malformed auricles, conductive deafness, downward obliquity of the palpebral fissures, lower eyelid coloboma, malar and mandibular hypoplasia, malocclusion and cleft palate. Mutations at the TCS locus lead to the pleiotropic effects primarily involving structures derived from the 1st & 2nd branchial arches. The overall goal of this proposal is to localize and isolate the gene responsible for TCS and characterize the mutations in this disorder. We have recently mapped the TCS locus to human chromosome region 5q31.3->33.3. We propose to refine the location of the gene responsible for TCS. Towards this end we will 1) obtain DNA sequences from this region by screening human bacteriophage, cosmid and yeast artificial chromosome libraries, 2) generate a genetic map of this region by linkage analysis with TCS families and polymorphisms associated with these sequences, and 3) develop a physical map of the sequences in this region by conventional and pulsed field gel electrophoreses. For DNA sequences that are tightly linked with the disease locus, candidate genes will be identified by 1) gross alterations in their gene structure, 2) CpG-rich islands, 3) conservation in other species, 4) altered mRNA in affected individuals, and 5) complementary RNA in normal craniofacial tissues. Southern and Northern blot hybridization and polymerase chain reaction (PCR) will be utilized in these analyses. The gene will be isolated by screening a cDNA library, and overlapping clones will be sequenced. The TCS cDNA will be amplified from affected individuals by PCR and searched for evidence of mutation using the non- denaturing single-strand separation technique, denaturing gradient gel electrophoresis, and chemical cleavage method.Amplified fragments exhibiting evidence of mutation will be sequenced. Gene mutations can be analyzed with respect to their phenotypic effects among affected individuals. Variability of phenotype can be evaluated with respect to 1) different mutations, 2) different modes of inheritance such as genomic imprinting and gonadal mosaicism, and 3) evidence for allelic conditions. These studies will contribute to our understanding of craniofacial development and aid in diagnosis and counseling.